The 65 kilodalton DNA binding protein (65K DBP) of herpes simplex virus type 1 (HSV-l) has been shown to be encoded by UL42 and is one of only seven viral genes required for origin-dependent DNA replication. A temperature sensitive (ts) mutant containing a lesion in the 65K DBP gene does not synthesize viral DNA at the nonpermissive temperature, confirming the role of the 65K DBP in viral DNA replication in vivo. However, the exact function of the 65K DBP and how it interacts with the other replication proteins remains unknown and is the long term goal of this project. Due to its ability to encode so many of the proteins required for viral DNA replication, HSV provides a genetically tractable model system for studying DNA replication in a eukaryotic organism. Moreover, each of the seven replication genes, including the 65K DBP, are valid antiviral targets. In the short term, we will examine the general template specificity for DNA binding of the 65K DBP, and determine whether its ability to stimulate pol is due to template effects, increase in rate or processivity, increase in the ability of pol to cycle, or promotion of lagging strand synthesis. We will determine the various functional domains of the protein by determining which regions of the protein are specifically required for DNA binding, stimulation of the viral DNA polymerase (pol), nuclear localization, and origin-dependent DNA replication. Initially each of these domains will be studied using plasmids in which nested mutations in the 65K DBP gene have been engineered. For DNA binding and stimulation of pol, plasmids will be tran- scribed and translated in vitro to yield mutant protein. Transient transfection analysis of cells will be used to determine whether mutated plasmids express a 65K DBP capable of localizing in the nucleus and capable of supporting origin-dependent DNA synthesis. Once identified, domains will be further localized by site(oligo)- directed mutagenesis. A cell line will be constructed capable of supporting the growth of virus with defects in the 65K DBP gene and engineered mutations will be transferred into an intact viral genome. Isolated viral mutants will be tested for the effect of the mutation on the ability of the virus to replicate and synthesize viral DNA in vivo.